2,2'-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1.

The MDR-involved human GSTA1-1, an important isoenzyme overexpressed in several tumors leading to chemotherapeutic-resistant tumour cells, has been targeted by 2,2'-dihydroxybenzophenones and some of their carbonyl N-analogues, as its potential inhibitors. A structure-based library of the latter was built-up by a nucleophilic cleavage of suitably substituted xanthones to 2,2'-dihydroxy-benzophenones (5-9) and subsequent formation of their N-derivatives (oximes 11-13 and N-acyl hydrazones 14-16). Screening against hGSTA1-1 led to benzophenones 6 and 8, and hydrazones 14 and 16, having the highest inhibition potency (IC50 values in the range 0.18 ± 0.02 to 1.77 ± 0.10 µM). Enzyme inhibition kinetics, molecular modeling and docking studies showed that they interact primarily at the CDNB-binding catalytic site of the enzyme. In addition, the results from cytotoxicity studies with human colon adenocarcinoma cells showed low LC50 values for benzophenone 6 and its N-acyl hydrazone analogue 14 (31.4 ± 0.4 µM and 87 ± 1.9 µM, respectively), in addition to the strong enzyme inhibition profile (IC50(6)=1,77 ± 0.10 µM; IC50(14)=0.33 ± 0.05 µM). These structures may serve as leads for the design of new potent mono- and bi-functional inhibitors and pro-drugs against human GTSs.

Concluding the trilogy: The interaction of 2,2'-dihydroxy-benzophenones and their carbonyl N-analogues with human glutathione transferase M1-1 face to face with the P1-1 and A1-1 isoenzymes involved in MDR.

A series of 2,2'-dihydroxybenzophenones and their carbonyl N-analogues were studied as potential inhibitors against human glutathione transferase M1-1 (hGSTM1-1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1-1 within a range of 0-42% (25 µM). The IC50 values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 µΜ and 28.5 ± 2.5 µΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1-1 and hGSTA1-1 involved in MDR. All but one bind more strongly to A1-1, than M1-1 and P1-1, the latter being a poor binder. An order of potency A1-1 > > M1-1 >  P1-1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1-1. Enzyme kinetics with hGSTM1-1 (Km(CDNB) 213 ± 10 µΜ and Km(GSH) 303 ± 11 µΜ) revealed a competitive modality for 16 (Ki(16)  = 22.3 ± 1.1 µΜ) and a mixed one for 13 versus CDNB (Ki(13)  = 33.3 ± 1.6 µM for the free enzyme and Ki(13) ' = 17.7 ± 1.7 µM for the enzyme-CDNB complex). 5- or 5'-Bromo- or phenyl-substituted (but not in combination) inhibitors, having a H-bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1-1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.

Affinity chromatography matures as bioinformatic and combinatorial tools develop.

Affinity chromatography has the reputation of a more expensive and less robust than other types of liquid chromatography. Furthermore, the technique is considered to stand a modest chance of large-scale purification of proteinaceous pharmaceuticals. This perception is changing because of the pressure for quality protein therapeutics, and the realization that higher returns can be expected when ensuring fewer purification steps and increased product recovery. These developments necessitated a rethinking of the protein purification processes and restored the interest for affinity chromatography. This liquid chromatography technique is designed to offer high specificity, being able to safely guide protein manufactures to successfully cope with the aforementioned challenges. Affinity ligands are distinguished into synthetic and biological. These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated by three methods. The rational method features the functional approach and the structural template approach. The combinatorial method relies on the selection of ligands from a library of synthetic ligands synthesized randomly. The combined method employs both methods, that is, the ligand is selected from an intentionally biased library based on a rationally designed ligand. Biological ligands are selected by employing high-throughput biological techniques, e.g. phage- and ribosome-display for peptide and microprotein ligands, in addition to SELEX for oligonucleotide ligands. Synthetic mimodyes and chimaeric dye-ligands are usually designed by rational approaches and comprise a chloro-triazinlyl scaffold. The latter substituted with various amino acids, carbocyclic, and heterocyclic groups, generates libraries from which synthetic ligands can be selected. A 'lead' compound may help to generating a 'focused' or 'biased' library. This can be designed by various approaches, e.g.: (i) using a natural ligand-protein complex as a template; (ii) applying the principle of complementarity to exposed residues of the protein structure; and (iii) mimicking directly a natural biological recognition interaction. Affinity ligands, based on the peptide structure, can be peptides, peptide-mimetic derivatives (<30 monomers) and microproteins (e.g. 25-200 monomers). Microprotein ligands are selected from biological libraries constructed of variegated protein domains, e.g. minibody, Kunitz, tendamist, cellulose-binding domain, scFv, Cytb562, zinc-finger, SpA-analogue (Z-domain).

Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI-6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI-6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni-NTA agarose. Primary transformed plants harboring the gstI-6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.

Isoenzyme- and allozyme-specific inhibitors: 2,2'-dihydroxybenzophenones and their carbonyl N-analogues that discriminate between human glutathione transferase A1-1 and P1-1 allozymes.

The selectivity of certain benzophenones and their carbonyl N-analogues was investigated towards the human GSTP1-1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5-CT/TOPO-TA-hGSTP1*A, pOXO4-hGSTP1*B or pOXO4-hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1-1A 62% and 67%, 11 and 12 with P1-1C 51% and 70%, whereas that of 15 fell behind with P1-1B (41%). These findings were confirmed by IC50 values (74-125 µm). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (Ki(13/A)  = 63.6 ± 3.0 µm, Ki(15/B)  = 198.6 ± 14.3 µm, and Ki(11/C)  = 16.5 ± 2.7 µm). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1-1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity.

Engineering the xenobiotic substrate specificity of maize glutathione S-transferase I.

Glutathione S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of glutathione (GSH) to electrophilic sites on a variety of hydrophobic substrates. In the present study three amino acid residues (Trp12, Phe35 and Ile118) of the xenobiotic binding site (H-site) of maize GST I were altered in order to evaluate their contribution to substrate binding and catalysis. These residues are not conserved and hence may affect substrate specificity and/or product dissociation. The results demonstrate that these residues are important structural moieties that modulate an enzyme's catalytic efficiency and specificity. Phe35 and Ile118 also participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. The effect of temperature on the catalytic activity of the wild-type and mutant enzymes was also investigated. Biphasic Arrhenius and Eyring plots for the wild-type enzyme showed an apparent transition temperature at 35 degrees C, which seems to be the result of a change in the rate-limiting step of the catalytic reaction. Thermodynamic analysis of the activity data showed that the activation energy increases at low temperatures, whereas the entropy change seems to be the main determinant that contributes to the rate-limiting step at high temperatures.

Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis.

The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.

A portable fiber-optic pesticide biosensor based on immobilized cholinesterase and sol-gel entrapped bromcresol purple for in-field use.

A fiber-optic biosensor for the detection and determination of the pesticides carbaryl and dichlorvos was developed. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromcresol purple, deposited on an inner glass disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. Calibration curves were obtained for carbaryl and dichlorvos, with useful concentration ranges 0.11-8.0 mg l(-1) for carbaryl and 5.0-30 microg l(-1) for dichlorvos. The respective detection limits were 108 microg l(-1) and 5.2 microg l(-1). The method reproducibility was in the order of +/-3-5%. The method was successfully applied to the detection and determination of these pesticides in real water samples, without sample preparation steps. Recovery experiments were made and the accuracy of the method was 94.9%. No enzyme regeneration steps were applied and the sensor lifetime was 3 weeks (30% activity reduction). The bioactive mini sandwich can easily be replaced by simply unscrewing the terminal holding ring of the probe and placing a new sandwich, before the sensor is ready for use.

The interaction of the chemotherapeutic drug chlorambucil with human glutathione transferase A1-1: kinetic and structural analysis.

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

Designer xanthone: an inhibitor scaffold for MDR-involved human glutathione transferase isoenzyme A1-1.

Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC(50) values (7: 1.59 ± 0.25 µM, 8: 5.30 ± 0.30 µM, and 17: 8.56 ± 0.14 µM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 µM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC(50) = 10.54 ± 2.41 µM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC(50) = 151.3 ± 16.3 µM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs.

Synthesis and study of 2-(pyrrolesulfonylmethyl)-N-arylimines: a new class of inhibitors for human glutathione transferase A1-1.

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 µM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 µM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands.

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography.

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.

Kinetic analysis of maize glutathione S-transferase I catalysing the detoxification from chloroacetanilide herbicides.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of multi-functional enzymes involved in biodegradation of several herbicide classes. The ability of the maize isoenzyme GST I to detoxify from the acetanilide herbicide alachlor was investigated by steady-state kinetics and site-directed mutagenesis studies. Steady-state kinetics fit well to a rapid equilibrium random sequential bi-bi mechanism with intrasubunit modulation between GSH binding site (G-site) and electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent and thermodynamic data suggest that product release is rate-limiting. Three residues of GST I (Trp12, Phe35 and Ile118), which build up the xenobiotic binding site, were mutated and their functional and structural roles during alachlor conjugation were investigated. These residues are not conserved, hence may affect substrate specificity and/or product dissociation. The work showed that the key amino acid residue Phe35 modulates xenobiotic substrate binding and specificity, and participates in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. Trp12 and Ile118 do not seem to carry out such functions but instead, regulate the K(m) for alachlor by contributing to its productive orientation in the H-site. The results of the present work have practical significance since this may provide the basis for the rational design of new engineered GSTs with altered substrate specificity towards herbicides and may facilitate the design of new, more selective herbicides.